Re: Decal with heat
I presume you are talking about acid decalcifiers. Not only will the
proteins be hydrolyzed, but if you do not totally fix the bone, macerations
will occur and all will be lost. The H&E is one of the most important
stains for bone specimens, and acid decalcifiers already have damaging
effects on nuclear staining, you can't avoid them, so why add another
damaging factor with heat.
What kind of turn around time are you looking at?? on a femoral slab???
There are other ways to speed up large bone specimen decalcification in
acids and I would avoid heat at all costs.
You can increase the concentration of acid, formic acid from 4.5%
(Kristensens) to 15% and I have played with 20%, just treat stock formic
acid as though it is 100% formic acid (actually 88%) and use 15 mls in 85ml
water, calculate for 20%.
Hydrochloric acid decalcifiers are already ( approx 10 - 12% for commercial
mixtures) very concentrated, I never use HCl at more than 5%, I will dilute
stronger commercial mixtures or make up an 8% formic acid/4% hydrochloric
acid mixture, change daily and do endpoint testing faithfully ( you did not
say if you did this, chemically or with xray??)
Suspend bones in decalcifiers, I stir (this is questionable, but I do it to
disperse bubbles, keep bones from floating), separate bone slabs.
Make sure you reduce the size of bones, this alone speeds up
decalcification, get a good bone saw, and make sure your bones are fixed
100% in NBF to protect from the effects of acids.
At 09:58 AM 11/12/01 -0600, you wrote:
>I was wondering if anyone out there is decalcifying large bone specimens
>with heat (femoral heads for example)?
>According to literature, heat is not recommended since the loss of calcium
>salts occurs very fast and swelling and hydolysis of the bone occurs
>interfering with certain stains. But I'm sure there are ways around this,
>especially just for H&E staining.
> How is this being done? I would appreciate any information on this subject
>that would help accelerate the turn-around time on these
> Ruth Cazares
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610
406 994-4303 (FAX)
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